Background: Toxins are key virulence determinants of pathogens and can impair the function of host immune cells, including platelets. Insights into pathogen toxin interference with platelets will be pivotal to improve treatment of patients with bacterial bloodstream infections. Materials and Methods: In this study, we deciphered the effects of Staphylococcus aureus toxins α-hemolysin, LukAB, LukDE, and LukSF on human platelets and compared the effects with the pore forming toxin pneumolysin of Streptococcus pneumoniae. Activation of platelets and loss of platelet function were investigated by flow cytometry, aggregometry, platelet viability, fluorescence microscopy, and intracellular calcium release. Thrombus formation was assessed in whole blood.Results: α-hemolysin (Hla) is known to be a pore-forming toxin. Hla-induced calcium influx initially activates platelets as indicated by CD62P and αIIbβ3 integrin activation, but also induces finally alterations in the phenotype of platelets. In contrast to Hla and pneumolysin, S. aureus bicomponent pore-forming leukocidins LukAB, LukED, and LukSF do not bind to platelets and had no significant effect on platelet activation and viability. The presence of small amounts of Hla (0.2 µg/ml) in whole blood abrogates thrombus formation indicating that in systemic infections with S. aureus the stability of formed thrombi is impaired. Damage of platelets by Hla was not neutralized by intravenous immune globulins.

The human pathogen Staphylococcus aureus is considered mainly an extracellular, opportunistic pathogen, yet the bacterium is able to survive within and escape from host cells, including macrophages. An agr/ sae mutant of strain USA300 is unable to escape from human macrophages but can replicate and survive within macrophages. We questioned whether such “„non-toxic“” S. aureus resembles the less pathogenic coagulase-negative Staphylococcal species (CoNS) like S. carnosus, S. lugdunensis, S. capitis, S. warneri or S. pettenkoferi. We show that in contrast to the “„non-toxic“” S. aureus strains, the CoNS species are efficiently killed within 24 h post-infection in the macrophage-like THP-1 cells or in human primary macrophages. Bacterial persistence of “„non-toxic“” S . aureus or CoNS induced IL-1ß release but no cell-death. Mutations in genes coding for katalase, copprer transport or the regulatory system GraRS or SigB did not impact bacterial survival in THP-1 cells. Deletion of the superoxide dismutases sodA and sodM impaired S. aureus survival in human primary macrophages but not in THP-1 cells. However, expression of the S. aureus specific sodM in S. epidermidis was not sufficient to protect this species from being killed in THP-1 cells. Thus, at least in those cells better bacterial survival of S. aureus could not be linked to higher protection from ROS. However, “„non-toxic“” S. aureus was found to be insensitive to pH, whereas S. epidermidis was protected when phagosomal acidification was inhibited. Thus, species differences seem to be linked to different sensitivity to acidification.

Toxins are key virulence determinants of pathogens and can impair the function of host immune cells including platelets. Insights into pathogen toxin interference with platelets will be pivotal to improve treatment of patients with bacterial bloodstream infections. In this study, we deciphered the effects of Staphylococcus aureus toxins α-hemolysin, LukAB, LukDE and LukSF on human platelets and compared the effects with the pore forming toxin pneumolysin of Streptococcus pneumoniae. In contrast to pneumolysin, α-hemolysin initially activates platelets as indicated by CD62P and αIIbβ3 integrin expression, but the resulting pores also induce alterations in the phenotype of platelets and induce apoptosis of platelets. The presence of small amounts of α-hemolysin (0.2 µg/mL) in whole blood abrogates thrombus formation indicating that in systemic infections with S. aureus the stability of formed thrombi is impaired. This might be of high clinical relevance for S. aureus induced endocarditis of the aortic valves. Stabilizing the thrombi by inhibiting α-hemolysin induced impairment of platelets likely reduces the risk for septic (micro-)embolization. However, in contrast to pneumolysin, α-hemolysin induced platelets damage could not be neutralized by intravenous immune globulins. In contrast to α-hemolysin, S. aureus bi-component pore forming leukocidins LukAB, LukED and LukSF do not bind to platelets and had no significant effect on platelet activation and viability.Main point 1: α-hemolysin forms pores in platelets, which first activate but then result in apoptosis and impairs thrombus formation and stabilityMain point 2: Polyvalent immunoglobulins do not neutralize the mode of action of the toxin